HIV-1 envelope (Env) vaccines have failed to induce neutralizing antibodies (nabs) with strong activity against diverse HIV-1. However, the identification of potent, broad neutralizing monoclonal antibodies along with evidence that V2 and V3 loop antibodies contributed to protection in the RV144 vaccine trial, have increased optimism that an Env based vaccine is possible. However, it remains unclear how to present conserved Env epitopes on immunogens so that broadly active nabs are elicited. Native Env trimers in immune tissue are likely to be closed to protect critical sites from nabs. The trimer association domain (TAD) at the trimer apex along with residues in the CD4 binding loop region help maintain a closed conformation. We propose that understanding how TAD conformation is controlled will be critical for the development of trimeric Env immunogens that elicit broad and potent nabs. However, determinants that maintain a closed trimer and regulate TAD conformation are poorly understood. Here, we will use EMPIRIC (Exceedingly Meticulous and Parallel Investigation of Randomized Individual Codons), a novel saturation mutagenesis approach to identify residues in the V1V2 and V3 loops that regulate TAD conformations across clades. This approach is supported by strong preliminary data and will be used to identify mutant trimers that carry TADs with distinct conformations. Such trimers will represent novel immunogens for future studies. Aim 1. To identify amino acids in the TAD that influence viral replication using EMPIRIC. These amino acids are candidates for regulating TAD and trimer conformations: We will use EMPIRIC saturation mutagenesis to investigate V1, V2 and V3 loops of the TAD in clade A, B and C Envs. We will catalogue mutations in these Env regions that confer wild type or enhanced replication in PBMCs compared to wt Env+ virus, before analyzing their effects on Env and TAD conformation in aim 2. Aim 2. To characterize mutant Envs for modifications in TAD and trimer conformation: We will investigate how mutations identified in aim 1 impact on TAD and trimer conformation. We will use a novel trimer binding assay and Env+ pseudovirus neutralization tests to characterize the different Env mutants. A broad panel of monoclonal antibodies against the TAD, CD4bs and other Env regions as well as sCD4 will be tested to determine changes in TAD conformation and CD4bs exposure as well as effects on other Env sites. Our data will provide detailed information on TAD amino acids that regulate trimer conformation. We will create an Env structural map relating specific V1V2 abd V3 loop amino acids to (1) distinct conformations, (2) exposure of conserved and variable neutralization epitopes and (3) access for CD4. This information will help establish universal, cross clade rules for regulating TAD and trimer conformation that will be invaluable for design of next generation trimer immunogens aiming to elicit broad neutralizing antibodies.